tbuckley/cabot-search huggingface dataset:
Abstract 2527: Investigation of doxorubicin interference in flow cytometry measurements of apoptosis and necrosis
Authors: Rachel E. Nicoletto, Richard Wilkes, Clyde M. Ofner
Journal: Cancer Research (2023)

Abstract Background: Doxorubicin (DOX) is frequently used for investigations of breast cancer using flow cytometry with fluorescent stains for apoptosis and necrosis. DOX interference, however, can occur with these fluorescent measurements which is not reported in many published reports. The objectives of this study were to identify such interference and to assess its extent. Methods: MDA-MB-231 and MCF-7 cells were incubated for 24 h with 0.1, 1, and 10 µM DOX. Cells were then stained for apoptosis and necrosis using two commercial assay kits. Fluorescence was measured with FL1 (apoptosis), FL2 (primary for DOX), and FL3 (necrosis) detectors in cells with DOX alone, with DOX plus stains, and in control cells without DOX. Measurements were repeated two to five times using replicates and trials. Statistics were evaluated by t-tests. Results: Overall, DOX fluorescence interference increased with concentration in both cell lines. After DOX incubation, cells stained with caspase 3/7 and SYTOX showed fluorescence in the FL1 detector indicating caspase 3/7 activation in apoptosis. But the DOX component of fluorescence with this detector ranged from 11 to 95%. Dox fluorescence in the FL3 detector for SYTOX measurements indicating necrosis ranged from 26 to 96% of total fluorescence. Cells stained with Annexin V (with the FITC label) and propidium iodide (PI) showed fluorescence in the FL1 detector for Annexin V indicating apoptosis. However, the DOX component of fluorescence ranged from 11 to 25%. DOX fluorescence in the FL3 detector for PI indicating necrosis ranged from 87 to 106%. Conclusions: Without correction for DOX interference, and even at low concentrations, substantial errors can occur for apoptosis and necrosis measurements using flow cytometry with these stains in breast cancer cells. At high DOX concentrations, the interference can erroneously indicate an extensive presence of these cell death processes. Corrections for DOX interference include background subtraction and flow cytometer compensation. Acknowledgments: The authors gratefully acknowledge financial support from the Agnes Varis Trust for Women in Science (REN), the Milton Lev Memorial Faculty Research Fund (CMO) as well as Saint Joseph’s University support for student research. The authors also thank Bin Chen, PhD, and Richard Howley for assistance with flow cytometry. Citation Format: Rachel E. Nicoletto, Richard Wilkes, Clyde M. Ofner. Investigation of doxorubicin interference in flow cytometry measurements of apoptosis and necrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2527.
Abstract 1980: Integrating common and rare genetic variants to predict cancer risks in diverse populations
Authors: Abdulkadir Elmas, Hannah Kittrell, Kuan-lin Huang
Journal: Cancer Research (2023)

Abstract Inherited germline genetic factors contribute substantially to cancer risk. Large-scale DNA-Sequencing (DNA-Seq) studies have identified rare pathogenic variants in 5~10% of adult cancer cases, while genome-wide association studies (GWAS) have revealed hundreds of common variants that require aggregation using polygenic risk scores (PRS). However, rare and common genetic variants are often assessed separately and prohibit accurate risk assessments. Here, we developed methods to combine common and rare variants for improved cancer risk predictions using the diverse Mount Sinai BioMe population cohort of 49,854 individuals, including 22% African Americans, 1.9% Asian, 34% Latinx/Hispanic, 32% Europeans, and 11% of Other self-reported ancestries. By linking and analyzing EHR data, we identified over 4,000 cancer cases and another 815 individuals with family history, and analyzed 14 solid tumor types with at least 50 cases with pass-QC whole-exome sequencing (WES) data. The landscape of pathogenic variants differs across ancestries and highlighted multiple founder variants, while PRSs predict cancer risks in major cancer types across different ancestries. Notably, we detect significant interactions of common and rare genetic variants contributing to risks of selected cancer types. Machine learning models that combine both common and rare variants show higher predictive accuracy than using common or rare variants alone. Overall, our study underscores the importance of jointly considering common and rare genetic factors to provide accurate cancer risk assessment for diverse individuals. Citation Format: Abdulkadir Elmas, Hannah Kittrell, Kuan-lin Huang. Integrating common and rare genetic variants to predict cancer risks in diverse populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1980.
Abstract 228: Recurrent mitochondrial mutations in thyroid cancer
Authors: Julia F. Hopkins, Zoe Fleischmann, Dexter X. Jin, Jonathan Keith Killian, Meagan Montesion
Journal: Cancer Research (2023)

Abstract Despite the importance of mitochondrial function on the bioenergetics and metabolism of the cell, understanding the functional impact of somatic mitochondrial mutations has been difficult. Additionally, small cohorts, as used in previous studies, lack the power to estimate the prevalence of specific recurrent mutations. We evaluated 177540 advanced cancer patients, across 84 different tumor types, who were sequenced as part of routine clinical care on the FoundationOne®CDx platform. Off-target sequencing reads were mapped to the mitochondrial genome (rCRS) and variant calling was performed. We had a median read depth of 18 and filtering by allele frequency (&amp;lt;= 95% and &amp;gt;= 5%) led to the identification almost 400,000 predicted somatic mitochondrial variants. Over 80% of patients had at least one mitochondrial variant detected, with an average of 2.2 mitochondrial variants detected per patient. We next assessed each recurrent mitochondrial short variant to look for associations with specific cancers. The alteration with the strongest enrichment was G3224A, a mutation in the mitochondrial leucine tRNA gene (MT-TL1), which had a prevalence of 3.6% (51/1426) in thyroid cancer samples, as compared to 0.05% (85/176114) in all other disease types (OR=76.8, P&amp;lt;2.2e-16). The G3244A mutation has been previously identified as occurring in thyroid cancer, specifically Hurthle cell carcinomas, as well as in the mitochondrial disease MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). Thyroid cancer samples in our dataset with G3244A were found to be associated with thyroid follicular carcinomas (7/124, 5.6%) and Hurthle cell carcinomas (7/57, 12.2%). Additional recurrent MT-TL1 mutations were identified in the thyroid cancer cohort at positions 3242 (9/1426) and 3248 (5/1426). We also identified a mutation in the NADH dehydrogenase subunit MT-ND1. G3380A has been previously shown to occur in thyroid and colorectal cancer and results in a R25Q mutation that is thought to affect ND1 function. G3380A was identified in 26/1426 (1.8%) of thyroid cancer patients. The majority of G3380A variants occurred in thyroid anaplastic carcinomas 23/26 (88.5%). Our dataset has allowed us to identify and determine the prevalence of rare mitochondrial short variant alterations in cancer. Further research is needed to investigate associations of these alterations with other clinical features. Citation Format: Julia F. Hopkins, Zoe Fleischmann, Dexter X. Jin, Jonathan Keith Killian, Meagan Montesion. Recurrent mitochondrial mutations in thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 228.
Abstract 3715: Pharmacological targeting of circadian clock genes reveals regulatory mechanisms of E-box regulated genes in cancer
Authors: Yuanzhong Pan, Heinz-Josef Lenz, Evanthia Roussos Torres, Steve A. Kay
Journal: Cancer Research (2023)

Abstract Transcription factors (TFs) are key regulators of homeostasis and cancer. Recent advances in pharmacological targeting of TFs makes them valuable targets in various diseases, but systems-level understanding of the function and regulation of TFs from different families in cancers remains lacking. Our lab and collaborators have been developing new small molecules to modulate the functions of key regulators of the circadian clock BMAL1 and CLOCK, which are bHLH family TFs that activate gene expression by binding to a DNA motif called E-box, with a consensus sequence of CANNTG. E-boxes are ubiquitous in the genome and broadly regulate essential physiological processes of the cell, but their molecular biology in cancer is largely unknown. In the current work we tested the ability of small molecules to regulate the expression of BMAL1 and CLOCK target genes. Across different cell lines of various cancer types, we found that stabilization of one of the negative regulators of CLOCK, CRY2, by our novel compound called SHP1705, is the most consistent to suppress canonical BMAL1 target genes. GO and GSEA analysis of RNA-seq data shows that SHP1705 also significantly changed most core regulators of circadian rhythms as well as other E-box-binding proteins. However, SHP1705 has IC50 values above 10µM in most tested cells, creating a challenge for efficacy in a clinical setting. Because BMAL1 requires the proteasome to sustain the turnover of a transcription burst, we hypothesized proteasome inhibitors (PI) MG132 and carfilzomib may synergize with SHP1705. Consequently, we found strong synergy with both PIs. Analysis of TF-target genes in combination-treated cells reveals significant changes in expression profiles, suggesting that these genes are regulated by E-box, and other related/alternative promoter elements: CCAAT-box, CG-rich motifs, and ATGGC. Co-enrichment of these elements revealed the components of E-box containing promoters and showed potential properties of E-box-regulated gene expression. Co-existence of these motifs in the same proximal promoter also potentially allows RNAPII multiple choices of alternative promoters and could promote expression of variants in cancer cells. Our results support the necessity of inhibiting the whole promoter and provide an example of how to approach designer pharmacological strategies to achieve this purpose. Citation Format: Yuanzhong Pan, Heinz-Josef Lenz, Evanthia Roussos Torres, Steve A. Kay. Pharmacological targeting of circadian clock genes reveals regulatory mechanisms of E-box regulated genes in cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3715.
Abstract 91: Multi-omic single-cell approach reveals transcriptional and epigenetic plasticity in breast cancer
Authors: Hisham Mohammed
Journal: Cancer Research (2023)

Abstract Breast cancer is known to be highly heterogenous at the transcriptional and genetic level. In order to study functional consequences of this heterogeneity, we applied single-cell RNA-seq and single-cell Multi-ome (RNA+ATAC) on hormone responsive cell line and organoid models after multiple timepoint hormone treatments. We also developed TITAN, an LDA based machine learning tool to identify ‘cell states’ in single-cell datasets. Our results suggest that response to hormone signalling is highly heterogenous at the single-cell level, resulting in multiple divergent epigenetic and transcriptional cell states. We identified driver transcription factors for these states and defined epigenetic mechanisms regulating state change. Subsequently, we also investigated epigenetic and transcriptional states in primary breast tumors. We analysed 20 breast tumor samples using the single-cell Multi-ome (ATAC+RNA) approach and compared them to matched adjacent normal tissues. Our results identified epigenetic reprogramming events in breast cancer and novel transcription factors in the process. Using bulk DNA-sequencing as a reference, we inferred sub-clonal populations from both the RNA and ATAC modalities, allowing us to define heterogenous chromatin accessibility events that align with genetic sub-clones as well as those that are independent of it. Our dataset provides a comprehensive view of epigenetic and transcriptional events in breast tumorigenesis. Citation Format: Hisham Mohammed. Multi-omic single-cell approach reveals transcriptional and epigenetic plasticity in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 91.
Abstract 1077: Porcupine inhibition enhances enzalutamide efficacy in drug resistant prostate cancer
Authors: Katelyn Makayla Jones, Xiaoqi Liu
Journal: Cancer Research (2023)

Abstract Androgen receptor (AR) signaling continues to participate as a vital component of castration-resistant prostate cancer (CRPC). Subsequently, this has led to the development of Androgen Signaling Inhibitors (ASI), specifically Enzalutamide (ENZ), which is a direct inhibitor of AR, to clinically manage CRPC. Inevitably, ENZ treatment only provides improvement for approximately two months before advancing to an incurable form, ENZ-resistant CRPC. With PCa ranking as the second leading cause of cancer-related deaths in USA males, there is an urgency and necessity for the discovery and development of novel therapeutic approaches for CRPC. Wnt signaling has been extensively documented in its involvement in PCa and the tumor microenvironment (TME), however the mechanism of how the Wnt signaling cascades contribute to ENZ resistance is still ambiguous. Recently we have published that the activation of the canonical Wnt pathway contributes to the progression of ENZ resistance in CRPC and using a combination of β-catenin inhibitor with ENZ resulted in the synergistic inhibition of patient derived xenograft (PDX) tumor growth. Regarding the non-canonical Wnt pathway, we confirmed its contribution to invasion and migration which leads to metastasis in ENZ-resistant CRPC, and when the downstream effector ROCK is depleted or depleted ROCK cells are treated with ENZ, there is a significant hindering of cell migration and invasion. Also, utilizing a combination therapy of ROCK inhibitor with ENZ synergistically inhibited the growth of PDX tumors. Hence the reasoning that by simultaneously inhibiting both the canonical and non-canonical Wnt signaling cascade will result in the inhibition of cell proliferation, migration, and invasion. The goal of this study was to define the mechanism of PORCN in ENZ-resistant CRPC and develop a therapeutic approach to combat this disease. My research has determined that Porcupine (PORCN) is associated with CRPC progression to ENZ-resistance, and that an inhibition or loss of PORCN has resulted in the regain of ENZ sensitivity in ENZ-resistant models. This model has also demonstrated that PORCN and Wnt signaling engage in a paramount role contributing to AR activation, promoting CRPC progression, and the development of ENZ resistance. Citation Format: Katelyn Makayla Jones, Xiaoqi Liu. Porcupine inhibition enhances enzalutamide efficacy in drug resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1077.
Abstract 6050: Analysis of diverse metabolic pathways using bioluminescent cell-based assays
Authors: Donna Leippe, Natasha Karassina, Mike Valley, Kayla Sylvester, Jolanta Vidugiriene
Journal: Cancer Research (2023)

Abstract Cellular energy metabolism is a complex system of many interconnected metabolic pathways that govern both cell health and function. Cancer cells are adept at quickly switching between metabolic pathways to adapt to nutrient limitations in the environment, enhancing their growth advantages while negatively impacting other cells in the tumor microenvironment such as immune cells. To facilitate metabolic characterization of cancer cells, we developed assays for detection of metabolites involved in key pathways, such as glycolysis and glutaminolysis, using a bioluminescent NADH detection technology coupled to specific dehydrogenases. We have now extended these assays to include metabolites involved in glycogen, lipid, ketone body and amino acid metabolism, as well as intermediates in the TCA cycle such as malate, isocitrate, and 2-hydroxyglutarate. A second core bioluminescent hydrogen peroxide detection system was also used to develop an assay for pyruvate. Here we describe the use of these assays to measure metabolite levels in cell lysates and cell culture media collected from cancer cell lines plated in 96- or 384-well plates and in 2D or 3D cultures. Changes in metabolite levels were monitored in response to altered media composition and drug treatments. Levels were also compared between different cell lines. In comparative studies of glioblastoma cell lines, 2-hydroxyglutarate production was higher in cells expressing mutant isocitrate dehydrogenase. By reconfiguring the NADH detection technology to measure cellular enzyme activity rather than metabolite levels, we were able to detect activity of dehydrogenases that participate in the glycolysis pathway, the TCA cycle, and pentose phosphate pathway. Assays for cellular malate dehydrogenase, isocitrate dehydrogenase and other dehydrogenase enzymes were sensitive, requiring ≤ 400 lysed cells per assay. The expanded collection of bioluminescent assays provides a way to quickly characterize the metabolic states and behaviors of cancer cells to help identify pathways that may present therapeutic targets. Citation Format: Donna Leippe, Natasha Karassina, Mike Valley, Kayla Sylvester, Jolanta Vidugiriene. Analysis of diverse metabolic pathways using bioluminescent cell-based assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6050.
Abstract 1079: Combination of tipifarnib with KRAS G12C inhibitors to prevent adaptive resistance
Authors: Hetika Vora Patel, Alison Smith, Stacia Chan, Linda Kessler, Francis Burrows, Shivani Malik
Journal: Cancer Research (2023)

Abstract Selective KRAS G12C inhibitors, including AMG510 (sotorasib) and MRTX849 (adagrasib), have exhibited clinical activity in patients with non-small cell lung cancer (NSCLC); however, drug resistance and relapse inevitably occur through various mechanisms, which limits the ability of these therapeutic agents to achieve durable responses. Resistance mechanisms include feedback reactivation of compensatory, oncogenic signaling pathways and the attainment of a drug-tolerant state, each of which allows cancer cells to survive the anti-tumor effects of these highly active drugs. Compensatory mechanisms ameliorating KRAS blockade include HRAS/NRAS and the PI3K-AKT-mTOR pathway. Tipifarnib is a clinical stage farnesyltransferase inhibitor known to inhibit multiple farnesylation-dependent proteins in tumor cells. We have recently shown that tipifarnib can prevent feedback-mediated adaptive resistance to the PI3Kα inhibitor, alpelisib, through inhibition of both HRAS and RHEB, allowing simultaneous blockade of two key nodes of the oncogenic MAPK- and PI3K-signaling pathways. In another example, tipifarnib has been demonstrated by others to inhibit the ability of tumor cells to enter a drug-tolerant state induced by the EGFR inhibitor, osimertinib, in EGFR-mutant NSCLC models. In these models, inhibition of farnesylation of certain RHO proteins appear to contribute to the death of drug-tolerant cells prior to the emergence of acquired resistance mutations. Building on these two mechanisms of how tipifarnib may prevent adaptive resistance, we propose that tipifarnib can be used as an effective partner drug to delay or prevent the onset of adaptive resistance to KRAS G12C inhibitors. We have conducted in vitro 2D and 3D viability and regrowth experiments using combinations of tipifarnib with KRAS G12C inhibitors and have observed synergistic, anti-proliferative effects in KRAS G12C NSCLC cell lines as well as enhanced activity of combination in a KRAS G12C NSCLC PDX model. We are currently expanding the scope of in vitro and in vivo combination studies to further evaluate the molecular mechanism(s) of resistance that tipifarnib targets when combined with KRAS G12C inhibitors. Citation Format: Hetika Vora Patel, Alison Smith, Stacia Chan, Linda Kessler, Francis Burrows, Shivani Malik. Combination of tipifarnib with KRAS G12C inhibitors to prevent adaptive resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1079.